Histone deacetylase inhibitors based on trihalomethylcarbonyl compounds

ABSTRACT

Histone deacetylase is a metallo-enzyme with zinc at the active site. Compounds having a zinc-binding moiety, for example, a trihalomethylcarbonyl group, such as a trifluoromethylcarbonyl group, can inhibit histone deacetylase. Histone deacetylase inhibition can repress gene expression, including expression of genes related to tumor suppression. Accordingly, inhibition of histone deacetylase can provide an alternate route for treating cancer, hematological disorders, e.g., hemoglobinopathies, autosomal dominant disorders, e.g. spinal muscular atrophy and Huntington&#39;s disease, genetic related metabolic disorders, e.g., cystic fibrosis and adrenoleukodystrophy, or for stimulating hematopoietic cells ex vivo.

CLAIM OF PRIORITY

[0001] This application claims priority under 35 USC §119(e) to U.S.patent application Ser. No. 60/382,075, filed on May 22, 2002, theentire contents of which is hereby incorporated by reference.

TECHNICAL FIELD

[0002] This invention relates to trihalomethylcarbonyl compounds, andmore particularly to trihalomethylcarbonyl compounds that are histonedeacetylase inhibitors.

BACKGROUND

[0003] DNA in the nucleus of the cell exists as a hierarchy of compactedchromatin structures. The basic repeating unit in chromatin is thenucleosome. The nucleosome consists of a histone octamer of proteins inthe nucleus of the cell around which DNA is wrapped twice. The orderlypackaging of DNA in the nucleus plays an important role in thefunctional aspects of gene regulation. Covalent modifications of thehistones have a key role in altering chromatin higher order structureand function and ultimately gene expression. The covalent modificationof histones, such as acetylation, occurs by enzymatically mediatedprocesses.

[0004] Regulation of gene expression through the inhibition of thenuclear enzyme histone deacetylase (HDAC) is one of several possibleregulatory mechanisms whereby chromatin activity can be affected. Thedynamic homeostasis of the nuclear acetylation of histones can beregulated by the opposing activity of the enzymes histone acetyltransferase (HAT) and histone deacetylase (HDAC). Transcriptionallysilent chromatin can be characterized by nucleosomes with low levels ofacetylated histones. Acetylation reduces the positive charge ofhistones, thereby expanding the structure of the nucleosome andfacilitating the interaction of transcription factors with the DNA.Removal of the acetyl group restores the positive charge, condensing thestructure of the nucleosome. Histone acetylation can activate DNAtranscription, enhancing gene expression. Histone deacetylase canreverse the process and can serve to repress gene expression. See, forexample, Grunstein, Nature 389, 349-352 (1997); Pazin et al., Cell 89,325-328 (1997); Wade et al., Trends Biochem. Sci. 22, 128-132 (1997);and Wolffe, Science 272, 371-372 (1996).

SUMMARY

[0005] Histone deacetylase is a metallo-enzyme with zinc at the activesite. Compounds having a zinc-binding moiety, for example, atrihalomethylcarbonyl group such as a trifluoromethylcarbonyl group, caninhibit histone deacetylase. Histone deacetylase inhibition can altergene expression, including expression of genes related to tumorsuppression. Accordingly, inhibition of histone deacetylase can providean alternate route for treating cancer, hematological disorders, e.g.,hemoglobinopathies, genetic related metabolic disorders, e.g., cysticfibrosis and adrenoleukodystrophy, autosomal dominant disorders, e.g.Huntington's disease and spinal muscular atrophy, or for stimulatinghematopoietic cells ex vivo.

[0006] In one aspect, a compound has the formula (I):

[0007] In another aspect, method of inhibiting histone deacetylationactivity in cells includes contacting the cells with an effective amountof a compound containing a trihalomethylcarbonyl group, thereby treatingone or more disorders mediated by histone deacetylaseor stimulatinghematopoietic cells ex vivo, and determining whether the level ofacetylated histones in the treated cells is higher than in untreatedcells under the same conditions. The compound can be of formula (I). Inthe method, the compound can be 1,1,1-trifluoro-5-phenyl-2-pentanone,1,1,1-trifluoro-8-phenyl-3,5,7-octatrien-2-one, or1,1,1-trifluoro-8-phenoxy-3,5,7-octatrien-2-one. The disorder can becancer, hemoglobinopathies, thalassemia, sickle cell anemia, cysticfibrosis, protozoan infection, spinal muscular atrophy, Huntington'sdisease, alpha-1 anti-trypsin, retrovirus gene vector reactivation,wound healing, hair growth, peroxisome biogenesis disorder, oradrenoleukodystrophy.

[0008] In formula (I), A is a cyclic moiety selected from the groupconsisting of C₃₋₁₄ cycloalkyl, 3-14 membered heterocycloalkyl, C₄₋₁₄cycloalkenyl, 3-8 membered heterocycloalkenyl, aryl, or heteroaryl. Thecyclic moiety is optionally substituted with alkyl, alkenyl, alkynyl,alkoxy, hydroxyl, hydroxylalkyl, halo, haloalkyl, amino, thio,alkylthio, arylthio, aralkylthio, acylthio, alkylcarbonyloxy,alkyloxycarbonyl, alkylcarbonyl, alkylsulfonylamino, aminosulfonyl, oralkylsulfonyl. Alternatively, A is a saturated branched C₃₋₁₂hydrocarbon chain or an unsaturated branched C₃₋₁₂ hydrocarbon chainoptionally interrupted by —O—, —S—, —N(R^(a))—, —C(O)—, —N(R^(a))—SO₂—,—SO₂—N(R^(a))—, —N(R^(a))—C(O)—O—, —O—C(O)—N(R^(a))—,—N(R^(a))—C(O)—N(R^(b))—, —O—C(O)—, —C(O)—O—, —O—SO₂—, —SO₂—O—, or—O—C(O)—O—. Each of R^(a) and R^(b), independently, can be hydrogen,alkyl, alkenyl, alkynyl, alkoxy, hydroxylalkyl, hydroxyl, or haloalkyl.Each of the saturated and the unsaturated branched hydrocarbon chain isoptionally substituted with alkyl, alkenyl, alkynyl, alkoxy, hydroxyl,hydroxylalkyl, halo, haloalkyl, amino, thio, alkylthio, arylthio,aralkylthio, acylthio, alkylcarbonyloxy, alkyloxycarbonyl,alkylcarbonyl, alkylsulfonylamino, aminosulfonyl, or alkylsulfonyl.

[0009] In formula (I), each of Y¹ and Y², independently, is —O—, —S—,—N(R^(c))—, —N(R^(c))—C(O)—O—, —N(R^(c))—C(O)—, —C(O)—N(R^(c))—,—O—C(O)—N(R^(c))—, —N(R^(c))—C(O)—N(R^(d))—, —O—C(O)—O—, or a bond. Eachof R^(c) and R^(d), independently, can be hydrogen, alkyl, alkenyl,alkynyl, alkoxy, hydroxylalkyl, hydroxyl, or haloalkyl.

[0010] In formula (I), L is a straight C₃₋₁₂ hydrocarbon chainoptionally containing at least one double bond, at least one triplebond, or at least one double bond and one triple bond. The hydrocarbonchain can be optionally substituted with C₁₋₄ alkyl, C₂₋₄ alkenyl, C₂₋₄alkynyl, C₁₋₄ alkoxy, hydroxyl, halo, amino, thio, alkylthio, arylthio,aralkylthio, acylthio, nitro, cyano, C₃₋₅ cycloalkyl, 3-5 memberedheterocycloalkyl, monocyclic aryl, 5-6 membered heteroaryl, C₁₋₄alkylcarbonyloxy, C₁₋₄ alkyloxycarbonyl, C₁₋₄ alkylcarbonyl, or formyl.The hydrocarbon chain can be optionally interrupted by —O—, —N(R^(e))—,—N(R^(e))—C(O)—O—, —O—C(O)—N(R^(e))—, —N(R^(e))—C(O)—N(R^(f))—, or—O—C(O)—O—. Each of R^(e) and R^(f), independently, can be hydrogen,alkyl, alkenyl, alkynyl, alkoxy, hydroxylalkyl, hydroxyl, or haloalkyl.

[0011] In formula (I), X¹ is O or S.

[0012] In formula (I), X² is a halogen.

[0013] In formula (I), when Y¹ and Y² are each a bond, L is a C₆₋₁₂hydrocarbon chain containing at least one double bond at C1, C2, C3 orC5 of the hydrocarbon chain from C═X¹, at least one triple bond, or atleast one double bond and one triple bond.

[0014] In certain circumstances: X² can be fluorine; Y¹ can be not abond; each of Y¹ and Y², independently, can be —O—, —N(R^(c))—, or abond; X¹ can be O; L can be a C₆₋₈ hydrocarbon chain substituted withC₁₋₂ alkyl, C₁₋₂ alkoxy, hydroxyl, —NH₂, —NH(C₁₋₂ alkyl), or —N(C₁₋₂alkyl)₂; L can be a C₄₋₁₂ hydrocarbon chain; L can be a C₅₋₁₂hydrocarbon chain; L can be a C₅₋₁₀ hydrocarbon chain, L can be anunsaturated hydrocarbon chain containing at least two double bonds; or Acan be a C₅₋₈ cycloalkenyl, 5-8 membered heteroalkenyl, phenyl,naphthyl, indanyl, or tetrahydronaphthyl optionally substituted withalkyl alkenyl, alkynyl, alkoxy, hydroxyl, hydroxylalkyl, halo,haloalkyl, or amino.

[0015] In certain circumstances, A is phenyl, Y¹ is a bond, and L is aC₆₋₁₂ hydrocarbon chain containing three double bonds and the carbonadjacent to Y¹ is substituted with phenyl. In other circumstances, A isphenyl, Y¹ is a bond, and L is a C₃₋₁₂ hydrocarbon chain and the carbonadjacent to Y¹ is substituted with two phenyls.

[0016] In formula (I), the compound can be1,1,1-trifluoro-8-phenyl-3,5,7-octatrien-2-one or1,1,1-trifluoro-8-phenoxy-3,5,7-octatrien-2-one.

[0017] A salt of any of the compounds can be prepared. For example, apharmaceutically acceptable salt can be formed when an amino-containingcompound of this invention reacts with an inorganic or organic acid.Some examples of such an acid include hydrochloric acid, hydrobromicacid, hydroiodic acid, sulfuric acid, phosphoric acid,p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid,benzoic acid, and acetic acid. Examples of pharmaceutically acceptablesalts thus formed include sulfate, pyrosulfate bisulfate, sulfite,bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate,metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate,propionate, decanoate, caprylate, acrylate, formate, isobutyrate,caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate,sebacate, fumarate, and maleate. A compound of this invention may alsoform a pharmaceutically acceptable salt when a compound of thisinvention having an acid moiety reacts with an inorganic or organicbase. Such salts include those derived from inorganic or organic bases,e.g., alkali metal salts such as sodium, potassium, or lithium salts;alkaline earth metal salts such as calcium or magnesium salts; orammonium salts or salts of organic bases such as morpholine, piperidine,pyridine, dimethylamine, or diethylamine salts.

[0018] It should be recognized that a compound of the invention cancontain chiral carbon atoms. In other words, it may have optical isomersor diastereoisomers.

[0019] Alkyl is a straight or branched hydrocarbon chain containing 1 to10 (preferably, 1 to 6; more preferably 1 to 4) carbon atoms. Examplesof alkyl include, but are not limited to, methyl, ethyl, propyl,isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl,2-methylhexyl, and 3-ethyloctyl.

[0020] Alkenyl and alkynyl refer to a straight or branched hydrocarbonchain containing 2 to 10 carbon atoms and one or more (preferably, 1-4or more preferably 1-2) double or triple bonds, respectively. Someexamples of alkenyl and alkynyl are allyl, 2-butenyl, 2-pentenyl,2-hexenyl, 2-butynyl, 2-pentynyl, and 2-hexynyl.

[0021] Cycloalkyl is a monocyclic, bicyclic or tricyclic alkyl groupcontaining 3 to 14 carbon atoms. Some examples of cycloalkyl arecyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, andnorbornyl. Heterocycloalkyl is a cycloalkyl group containing at leastone heteroatom (e.g., 1-3) such as nitrogen, oxygen, or sulfur. Thenitrogen or sulfur may optionally be oxidized and the nitrogen mayoptionally be quaternized. Examples of heterocycloalkyl includepiperidinyl, piperazinyl, tetrahydropyranyl, tetrahydrofuryl, andmorpholinyl. Cycloalkenyl is a cycloalkyl group containing at least one(e.g., 1-3) double bond. Examples of such a group include cyclopentenyl,1,4-cyclohexa-di-enyl, cycloheptenyl, and cyclooctenyl groups. By thesame token, heterocycloalkenyl is a cycloalkenyl group containing atleast one heteroatom selected from the group of oxygen, nitrogen orsulfur.

[0022] Aryl is an aromatic group containing a 5-14 member ring and cancontain fused rings, which may be saturated, unsaturated, or aromatic.Examples of an aryl group include phenyl, naphthyl, biphenyl,phenanthryl, and anthracyl. If the aryl is specified as “monocyclicaryl,” if refers to an aromatic group containing only a single ring,i.e., not a fused ring.

[0023] Heteroaryl is aryl containing at least one (e.g., 1-3) heteroatomsuch as nitrogen, oxygen, or sulfur and can contain fused rings. Someexamples of heteroaryl are pyridyl, furanyl, pyrrolyl, thienyl,thiazolyl, oxazolyl, imidazolyl, indolyl, benzofuranyl, andbenzthiazolyl.

[0024] The cyclic moiety can be a fused ring formed from two or more ofthe just-mentioned groups. Examples of a cyclic moiety having fusedrings include fluorenyl, dihydro-dibenzoazepine, dibenzocycloheptenyl,7H-pyrazino [2,3-c]carbazole, or 9,10-dihydro-9,10-[2]buteno-anthracene.

[0025] Amino protecting groups and hydroxy protecting groups arewell-known to those in the art. In general, the species of protectinggroup is not critical, provided that it is stable to the conditions ofany subsequent reaction(s) on other positions of the compound and can beremoved without adversely affecting the remainder of the molecule. Inaddition, a protecting group may be substituted for another aftersubstantive synthetic transformations are complete. Examples of an aminoprotecting group include, but not limited to, carbamates such as2,2,2-trichloroethylcarbamate or tertbutylcarbamate. Examples of ahydroxyl protecting group include, but not limited to, ethers such asmethyl, t-butyl, benzyl, p-methoxybenzyl, p-nitrobenzyl, allyl, trityl,methoxymethyl, 2-methoxypropyl, methoxyethoxymethyl, ethoxyethyl,tetrahydropyranyl, tetrahydrothiopyranyl, and trialkylsilyl ethers suchas trimethylsilyl ether, triethylsilyl ether, dimethylarylsilyl ether,triisopropylsilyl ether and t-butyldimethylsilyl ether; esters such asbenzoyl, acetyl, phenylacetyl, formyl, mono-, di-, and trihaloacetylsuch as chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl;and carbonates including but not limited to alkyl carbonates having fromone to six carbon atoms such as methyl, ethyl, n-propyl, isopropyl,n-butyl, t-butyl; isobutyl, and n-pentyl; alkyl carbonates having fromone to six carbon atoms and substituted with one or more halogen atomssuch as 2,2,2-trichloroethoxymethyl and 2,2,2-trichloro-ethyl; alkenylcarbonates having from two to six carbon atoms such as vinyl and allyl;cycloalkyl carbonates having from three to six carbon atoms such ascyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl; and phenyl orbenzyl carbonates optionally substituted on the ring with one or moreC₁₋₆ alkoxy, or nitro. Other protecting groups and reaction conditionscan be found in T. W. Greene, Protective Groups in Organic Synthesis,(3rd, 1999, John Wiley & Sons, New York, N.Y.).

[0026] Note that an amino group can be unsubstituted (i.e., —NH₂),mono-substituted (i.e., —NHR), or di-substituted (i.e., —NR₂). It can besubstituted with groups (R) such as alkyl, cycloalkyl, heterocycloalkyl,aryl, heteroaryl, aralkyl, or heteroaralkyl. Halo refers to fluoro,chloro, bromo, or iodo.

[0027] Inhibition of a histone deacetylase in a cell is determined bymeasuring the level of acetylated histones in the treated cells andmeasuring the level of acetylated histones in untreated cells andcomparing the levels. If the level of histone acetylation in the treatedcells increases relative to the untreated cells, histone deacetylase hasbeen inhibited.

[0028] Some disorders or physiological conditions may be mediated byhyperactive histone deacetylase activity. A disorder or physiologicalcondition that is mediated by histone deacetylase refers to a disorderor condition wherein histone deacetylase plays a role in triggering theonset thereof. Examples of such disorders or conditions include, but notlimited to, cancer, hemoglobinopathies (e.g., thalassemia or sickle cellanemia), cystic fibrosis, protozoan infection, spinal muscular atrophy,Huntington's disease, alpha-1 anti-trypsin, retrovirus gene vectorreactivation, wound healing, hair growth, peroxisome biogenesisdisorder, and adrenoleukodystrophy.

[0029] Other features or advantages will be apparent from the followingdetailed description of several embodiments, and also from the appendedclaims.

DETAILED DESCRIPTION

[0030] The compounds of formula (I) can generally be prepared accordingto the following methods. The trihalomethylcarbonyl compounds can beprepared from an aldehyde as shown in Scheme A. In particular, thetrihalomethylcarbonyl compound can be prepared by reacting the aldehydein THF with an equal amount of acetic acid and piperidine followed byaddition of 1,1,1-trifluoroacetone at room temperature under nitrogen.

[0031] The carbon-carbon double bond can be selectively reduced orotherwise modified. The aldehyde can be prepared, for example, from acarboxylic acid-containing compound. These compound can be prepared byany method known in the art. For example, a compound having anunsaturated hydrocarbon chain between A and —C(═X¹)— can be preparedaccording to scheme B:

[0032] where L′ is a saturated or unsaturated hydrocarbon linker betweenA and —CH═CH— in a compound of the invention, and A and X¹ has the samemeaning as defined above. See Coutrot et al., Syn. Comm. 133-134 (1978).Briefly, butyllithium is added to an appropriate amount of anhydroustetrahydrofuran (THF) at a very low temperature (e.g., −65° C.). Asecond solution having diethylphosphonoacetic acid in anhydrous THF isadded dropwise to the stirred butyllithium solution at the same lowtemperature. The resulting solution is stirred at the same temperaturefor an additional 30-45 minutes which is followed by the addition of asolution containing an aromatic acrylaldehyde in anhydrous THF over 1-2hours. The reaction mixture is then warmed to room temperature andstirred overnight. It is then acidified (e.g., with HCl) which allowsthe organic phase to be separated. The organic phase is then dried,concentrated, and purified (e.g., by recrystallization) to form anunsaturated carboxylic acid.

[0033] Alternatively, a carboxylic acid-containing compound can beprepared by reacting an acid ester of the formula A-L′—C(═O)—O-loweralkyl with a Grignard reagent (e.g., methyl magnesium iodide) and aphosphorus oxychloride to form a corresponding aldehyde, which can befurther oxidized (e.g., by reacting with silver nitrate and aqueousNaOH) to form an unsaturated carboxylic acid.

[0034] Other types of carboxylic acid-containing compounds (e.g., thosecontaining a linker with multiple double bonds or triple bonds) can beprepared according to published procedures such as those described, forexample, in Parameswara et al., Synthesis, 815-818 (1980) and Denny etal., J. Org. Chem., 27, 3404 (1962). As to compounds wherein X¹ is S,they can be prepared according to procedures described in Sandler, S. R.and Karo, W., Organic Functional Group Preparations, Volume III(Academic Press, 1972) at pages 436-437. Additional synthetic methodscan be found in March, J. Advanced Organic Chemistry, 4^(th) ed., (WileyInterscience, 1992).

[0035] Note that appropriate protecting groups may be needed to avoidforming side products during the preparation of a compound of theinvention. For example, if the linker L′ contains an amino substituent,it can be first protected by a suitable amino protecting group such astrifluoroacetyl or tert-butoxycarbonyl prior to being treated withreagents such as butyllithium. See, e.g., T. W. Greene, supra, for othersuitable protecting groups.

[0036] A compound produced by the methods shown above can be purified byflash column chromatography, preparative high performance liquidchromatography, or crystallization.

[0037] A pharmaceutical composition including the compound describedabove can be used to inhibit histone deacetylase in cells and can beused to treat disorders associated with abnormal histone deacetylaseactivity. Some examples of these disorders are cancers (e.g., leukemia,lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer,cervical cancer, renal cancer, prostate cancer, and breast cancer),hematological disorders (e.g., hemoglobinopathies, thalassemia, andsickle cell anemia), autosomal disorders (e.g. Huntington's disease andspinal muscular atrophy), and genetic related metabolic disorders (e.g.,cystic fibrosis, spinal muscular atrophy, peroxisome biogenesisdisorder, alpha-1 anti-trypsin, and adrenoleukodystrophy). The compoundsdescribed above can also stimulate hematopoietic cells ex vivo,ameliorating protozoal parasitic infection, accelerate wound healing,and protecting hair follicles.

[0038] An effective amount is defined as the amount which is required toconfer a therapeutic effect on the treated patient, and is typicallydetermined based on age, surface area, weight, and condition of thepatient. The interrelationship of dosages for animals and humans (basedon milligrams per meter squared of body surface) is described byFreireich et al., Cancer Chemother. Rep. 50, 219 (1966). Body surfacearea may be approximately determined from height and weight of thepatient. See, e.g., Scientific Tables, Geigy Pharmaceuticals, Ardsley,New York, 537 (1970). An effective amount of a compound described hereincan range from about 1 mg/kg to about 300 mg/kg. Effective doses willalso vary, as recognized by those skilled in the art, dependent on routeof administration, excipient usage, and the possibility of co-usage,pre-treatment, or post-treatment, with other therapeutic treatmentsincluding use of other chemotherapeutic agents and radiation therapy.Other chemotherapeutic agents that can be co-administered (eithersimultaneously or sequentially) include, but not limited to, paclitaxeland its derivatives (e.g., taxotere), doxorubicin, L-asparaginase,dacarbazine, amascrine, procarbazine, hexamethylmelamine, mitoxantrone,and gemicitabine.

[0039] The pharmaceutical composition may be administered via theparenteral route, including orally, topically, subcutaneously,intraperitoneally, intramuscularly, and intravenously. Examples ofparenteral dosage forms include aqueous solutions of the active agent,in a isotonic saline, 5% glucose or other well-known pharmaceuticallyacceptable excipient. Solubilizing agents such as cyclodextrins, orother solubilizing agents well-known to those familiar with the art, canbe utilized as pharmaceutical excipients for delivery of the therapeuticcompounds. Because some of the compounds described herein can havelimited water solubility, a solubilizing agent can be included in thecomposition to improve the solubility of the compound. For example, thecompounds can be solubilized in polyethoxylated castor oil (CremophorEL®) and may further contain other solvents, e.g., ethanol. Furthermore,compounds described herein can also be entrapped in liposomes that maycontain tumor-directing agents (e.g., monoclonal antibodies havingaffinity towards tumor cells).

[0040] A compound described herein can be formulated into dosage formsfor other routes of administration utilizing conventional methods. Forexample, it can be formulated in a capsule, a gel seal, or a tablet fororal administration. Capsules may contain any standard pharmaceuticallyacceptable materials such as gelatin or cellulose. Tablets may beformulated in accordance with conventional procedures by compressingmixtures of a compound described herein with a solid carrier and alubricant. Examples of solid carriers include starch and sugarbentonite. Compounds of this invention can also be administered in aform of a hard shell tablet or a capsule containing a binder, e.g.,lactose or mannitol, a conventional filler, and a tableting agent.

[0041] The activities of a compound described herein can be evaluated bymethods known in the art, e.g., MTT(3-[4,5-dimehtythiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay,clonogenic assay, ATP assay, or Extreme Drug Resistance (EDR) assay. SeeFreuhauf, J. P. and Manetta, A., Chemosensitivity Testing in GynecologicMalignancies and Breast Cancer 19, 39-52 (1994). The EDR assay, inparticular, is useful for evaluating the antitumor and antiproliferativeactivity of a compound described herein. Cells are treated for four dayswith a compound. Both untreated and treated cells are pulsed withtritiated thymidine for 24 hours. Radioactivity of each type of cells isthen measured and compared. The results are then plotted to generatedrug response curves, which allow IC₅₀ values (the concentration of acompound required to inhibit 50% of the population of the treated cells)to be determined.

[0042] Histone deacetylase inhibitory activity can be measured based onprocedures described by Hoffmann et al., Nucleic Acids Res., 27,2057-2058 (1999). Briefly, the assay starts with incubating the isolatedhistone deacetylase enzyme with a compound of the invention, followed bythe addition of a fluorescent-labeled lysine substrate (contains anamino group at the side chain which is available for acetylation). HPLCis used to monitor the labeled substrate. The range of activity of eachtest compound is preliminarily determined using results obtained fromHPLC analyses. IC₅₀ values can then be determined from HPLC resultsusing different concentrations of compounds of this invention. Allassays are duplicated or triplicated for accuracy. The histonedeacetylase inhibitory activity can be compared with the increasedactivity of acetylated histone for confirmation.

[0043] Compounds of this invention are also evaluated for effects ontreating X-linked adrenoleukodystrophy (X-ALD), a peroxisomal disorderwith impaired very long-chain fatty acid (VLCFA) metabolism. In such anassay, cell lines derived from human primary fibroblasts and(EBV-transformed lymphocytes) derived from X-ALD patients grown on RPMIare employed. Tissue culture cells are grown in the presence or absenceof test compounds. For VLCFA measurements, total lipids are extracted,converted to methyl esters, purified by TLC and subjected to capillaryGC analysis as described in Moser et al., Technique in DiagnosticBiochemical Genetics: A Laboratory Manual (ed. A., H. F.) 177-191(Wiley-Liss, New York, 1991). C24:0 β-oxidation activity oflymphoclastoid cells are determined by measuring their capacity todegrade [1-¹⁴C]-C24:0 fatty acid to water-soluble products as describedin Watkins et al., Arch. Biochem. Biophys. 289, 329-336 (1991). Thestatistical significance of measured biochemical differences betweenuntreated and treated X-ALD cells can be determined by a two-tailedStudent's t-test.

[0044] Further, compounds of the present invention are evaluated fortheir effects in treating cystic fibrosis (CF). Since the initial defectin the majority of cases of CF is the inability of mutant CF protein(CFTR) to fold properly and exit the ER, compounds of the invention aretested to evaluate their efficacy in increasing the trafficking of theCF protein out of the ER and its maturation through the Golgi. Duringits biosynthesis, CFTR is initially synthesized as a nascent polypeptidechain in the rough ER, with a molecular weight of around 120 kDa (BandA). It rapidly receives a core glycosylation in the ER, giving it amolecular weight of around 140 kDa (Band B). As CFTR exits the ER andmatures through the Golgi stacks, its glycosylation is modified until itachieves a terminal mature glycosylation, affording it a molecularweight of around 170 kDa (Band C). Thus, the extent to which CFTR exitsthe ER and traverses the Golgi to reach the plasma membrane may bereflected in the ratio of Band B to Band C protein. CFTR isimmunoprecipitated from control cells, and cells exposed to testcompounds. Both wt CFTR and ΔF508 CFTR expressing cells are tested.Following lysis, CFTR is immunoprecipitated using various CFTRantibodies. Immunoprecipitates are then subjected to in vitrophosphorylation using radioactive ATP and exogenous protein kinase A.Samples are subsequently solubilized and resolved by SDS-PAGE. Gels arethen dried and subject to autoradiography and phosphor image analysisfor quantitation of Bands B and C are determined on a BioRad personalfix image station.

[0045] Furthermore, compounds of this invention can be used to treathomozygous β thalassemia, a disease in which there is inadequateproduction of β globin leading to severe anemia. See Collins et al.,Blood, 85(1), 43-49 (1995).

[0046] Still further, compounds of the present invention are evaluatedfor their use as antiprotozoal or antiparasitic agents. The evaluationcan be conducted using parasite cultures (e.g., Asexual P. falciparum).See Trager, W. & Jensen, J. B., Science 193, 673-675 (1976). Testcompounds are dissolved in dimethyl sulfoxide (DMSO) and added to wellsof a flat-bottomed 96-well microtitre plate containing human serum.Parasite cultures are then added to the wells, whereas control wellsonly contain parasite cultures and no test compound. After at least oneinvasion cycle, and addition of labeled hypoxanthine monohydrochloride,the level of incorporation of labeled hypoxanthine is detected. IC₅₀values can be calculated from data using a non-linear regressionanalysis.

[0047] The toxicity of a compound described herein is evaluated when acompound of the invention is administered by single intraperitoneal doseto test mice. After administration of a predetermined dose to threegroups of test mice and untreated controls, mortality/morbidity checksare made daily. Body weight and gross necropsy findings are alsomonitored. For reference, see Gad, S. C. (ed.), Safety Assessment forPharmaceuticals (Van Nostrand Reinhold, N.Y., 1995).

[0048] The following specific examples, which described syntheses,screening, and biological testing of various compounds of thisinvention, are therefore, to be construed as merely illustrative, andnot limitative of the remainder of the disclosure in any way whatsoever.All publications recited herein, including patents, are herebyincorporated by reference in their entirety.

EXAMPLES

[0049] Synthesis of 5-phenyl-2,4-pentadienal

[0050] To a cooled (0-5° C.) 927 mL of 1 M solution of phenyl magnesiumbromide in tetrahydrofuran was added dropwise a solution ofcrotonaldehyde (65.0 g) in 130 mL of anhydrous ether over a period of 2hours and 45 minutes. The reaction was stirred for an additional 45minutes and then warmed to room temperature. After four more hours ofstirring, saturated ammonium chloride aqueous solution (750 mL) wasadded to the reaction. The mixture was extracted with 750 mL of ethertwice. The combined extract was dried over anhydrous potassium carbonateand filtered. The solvent was evaporated to give 135.88 g (99.9%) of thedesired 1-phenyl-2-buten-1-ol as an oil which was used in the next stepwithout further purification.

[0051] 1-Phenyl-2-buten-1-ol (135.88 g) was dissolved in 2300 mL ofdioxane and treated with 2750 mL of dilute hydrochloric acid (2.3 mL ofconcentrated hydrochloric acid in 2750 mL of water) at room temperature.The mixture was stirred overnight and then poured into 4333 mL of etherand neutralized with 2265 mL of saturated aqueous sodium bicarbonate.The aqueous phase was extracted with 1970 mL of ether. The combinedextract was dried over anhydrous potassium carbonate. Evaporation of thesolvent followed by Kugelrohr distillation at 30° C. for 30 minutesafforded 131.73 g (96.8%) of the desired 4-phenyl-3-buten-2-ol as an oilwhich was used in the next step without further purification.

[0052] Dimethylformamide (DMF, anhydrous, 14 mL) was cooled to 0-5° C.and phosphorus oxychloride (8.2 mL) was added dropwise over a period of40 minutes. The resulting solution was added dropwise to a cooled (0-5°C.) solution of 4-phenyl-3-buten-2-ol (10 g) in 32 mL of anhydrous DMFover a period of an hour. The reaction mixture was warmed to roomtemperature over a 35-minute period and then gradually heated up to 80°C. over a period of 45 minutes. The reaction was stirred at 80° C. forthree hours and then cooled to 0-5° C. To the cooled reaction solutionwas added dropwise a solution of sodium acetate (40 g) in deionizedwater (100 mL) over a period of one hour. The mixture was then reheatedto 80° C., stirred at 80° C. for an additional 10 minutes, cooled downto room temperature and extracted with ether (100 mL) twice. Thecombined extract was washed with brine (100 mL), dried over anhydroussodium sulfate, filtered and concentrated under vacuum to yield 8.78 gof the desired 5-phenyl-2,4-pentadienal as a liquid which was used inthe next step without further purification. ¹H NMR (CDCl₃, 300 MHz), δ(ppm) 7.51 (m, 2H), 7.37 (m, 3H), 7.26 (m, 1H), 7.01 (m, 2H), 6.26 (m,1H). The synthesis is summarized in Scheme I.

[0053] Synthesis of 5-phenoxy-2,4-pentadienal

[0054] 2-Formylvinyl phenyl ether is prepared by treatingphenoxyacetaldehyde with formaldehyde and diethylamine hydrochloridesalt. The ether is then reacting with a solution ofdiethylphosphonoacetic acid and n-butyllithium in anhydroustetrahydrofuran (THF) to form 5-phenoxy-2,4-pentadienoic acid.5-Phenoxy-2,4-pentadienal is obtained by first converting the carboxylicacid to a Weinreb amide using oxalyl chloride followed byN,O-dimethylhydroxylamine. Subsequently, reduction of the Weinreb amidewith lithium aluminum hydride (LAH) in THF leads to the formation of5-phenoxy-2,4-pentadienal. The synthesis is summarized in Scheme II.

[0055] Synthesis of 1,1,1-trifluoro-8-phenyl-3,5,7-octatrien-2-one

[0056] 1,1,1-Trifluoro-8-phenyl-3,5,7-octatrien-2-one is prepared asoutlined in Scheme IIIA. 1,1,1-Trifluoro-8-phenyl-3,5,7-octatrien-2-oneis prepared by reacting 5-phenyl-2,4-pentadienal in THF with an equalamount of acetic acid and piperidine followed by addition of1,1,1-trifluoroacetone at room temperature under nitrogen.

[0057] Synthesis of 1,1,1-trifluoro-8-phenoxy-3,5,7-octatrien-2-one

[0058] 1,1,1-Trifluoro-8-phenoxy-3,5,7-octatrien-2-one is prepared in asimilar manner, as shown in Scheme IIIB.

[0059] Assays

[0060] Compounds selected from 1,1,1-trifluoro-5-phenyl-2-pentanone(prepared according to the procedure described in Biochim. Biophys. Acta(1999), 1420 (1-2), 45-56),1,1,1-trifluoro-8-phenyl-3,5,7-octatrien-2-one, and1,1,1-trifluoro-8-phenoxy-3,5,7-octatrien-2-one are used in the assaysdescribed below.

[0061] In vitro Efficacy Studies—Extreme Drug Resistance (EDR) Assay

[0062] The PC3 cell line is maintained in RPMI supplemented with 10%fetal calf serum and antibiotics. Cells are suspended in 0.12% soft agarin complete medium and plated (2,000 cells per well) in different drugconcentrations onto a 0.4% agarose underlayer in 24-well plates. Platingcalls on agarose underlayers supports the proliferation only of thetransformed cells, ensuring that the growth signal stems from themalignant component of the tumor.

[0063] All compounds are dissolved in DMSO to 200× stock solutions.Stock solutions are diluted to 20× working solutions using the tissueculture medium, then are serially diluted and added to the 24-wellplates. The initial range of concentrations is 1 micromolar to 200micromolar. No significant changes in pH of the culture medium areobserved under the above conditions. Diluent control wells contain PC3cells treated with DMSO, at the dilutions used for appropriate drugtreatment. All experimental points are represented by two separate wells(duplicates). Four wells containing tumor cells that are not treatedwith drugs serve as negative controls in each experiment.

[0064] Cells are incubated with drugs under standard culture conditionsfor 5 days. Cultures are pulsed with tritiated thymidine (³H-TdR, NewLife Science Products, Boston, Mass.) at 5 μCi per well for the last 48hours of the culture period. Cell culture plates are then heated to 90°C. to liquefy the agarose, and cells are harvested onto glass fiberfilters, which are then placed into counting vials containing liquidscintillation fluid. The radioactivity trapped on the filters is countedwith a Beckman scintillation counter. The fraction of surviving cells isdetermined by comparing ³H-TdR incorporation in treated (experimentalpoints) and untreated (negative control) wells. Microsoft Excel is usedto organize the raw data on EDR experiments, and the SigmaPlot programis utilized to generate drug response curves. All drug response curvesare approximated as sigmoidal equations (characteristic for typical drugresponse curves) to fit the data. IC₅₀ values are determined using theapproximated sigmoidal curves and expressed as μM.

[0065] Histone (Hyper)Acetylation Assay

[0066] The effect of a compound described herein on histone acetylationcan be evaluated in an assay using mouse erythroleukemia cells. Studiesare performed with the DS19 mouse erythroleukemia cells maintained inRPMI 1640 medium with 25 mM HEPES buffer and 5% fetal calf serum. Thecells are incubated at 37° C.

[0067] Histones are isolated from cells after incubation for periods of2 and 24 hours. The cells are centrifuged for 5 minutes at 2000 rpm inthe Sorvall SS34 rotor and washed once with phosphate buffered saline.The pellets are suspended in 10 mL lysis buffer (10 mM Tris, 50 mMsodium bisulfite, 1% Triton X-100, 10 mM magnesium chloride, 8.6%sucrose, pH 6.5) and homogenized with six strokes of a Teflon pestle.The solution is centrifuged and the pellet washed once with 5 mL of thelysis buffer and once with 5 mL 10 mM Tris, 13 mM EDTA, pH 7.4. Thepellets are extracted with 2×1 mL 0.25 N HCl. Histones are precipitatedfrom the combined extracts by the addition of 20 mL acetone andrefrigeration overnight. The histones are pelleted by centrifuging at5000 rpm for 20 minutes in the Sorvall SS34 rotor. The pellets arewashed once with 5 mL acetone and protein concentration are quantitatedby the Bradford procedure.

[0068] Separation of acetylated histones is usually performed with anacetic acid-urea polyacrylamide gel electrophoresis procedure.Resolution of acetylated H4 histones is achieved with 6.25 N urea and nodetergent as originally described by Panyim and Chalkley, Arch. Biochem.Biophys. 130, 337-346 (1969). 25 μg Total histones are applied to a slabgel which is run at 20 mA. The run is continued for a further two hoursafter the Pyronin Y tracking dye has run off the gel. The gel is stainedwith Coomassie Blue R. The most rapidly migrating protein band is theunacetylated H4 histone followed by bands with 1, 2, 3 and 4 acetylgroups which can be quantitated by densitometry. The procedure fordensitometry involves digital recording using the Alpha Imager 2000,enlargement of the image using the PHOTOSHOP program (Adobe Corp.) on aMACINTOSH computer (Apple Corp.), creation of a hard copy using a laserprinter and densitometry by reflectance using the Shimadzu CS9000Udensitometer. The percentage of H4 histone in the various acetylatedstates is expressed as a percentage of the total H4 histone.

[0069] The concentration of a compound of the invention required todecrease the unacetylated H4 histone by 50% (i.e., EC₅₀) can then bedetermined from data obtained using different concentrations of testcompounds.

[0070] Histone Deacetylation Assay

[0071] The determination of the inhibition of histone deacetylase bycompounds described herein is based upon the procedure described byHoffmann et al., Nucleic Acids Res. 27, 2057-2058 (1999). The histonedeacetylase is isolated from rat liver as previously described in Kolle,D. et al. Methods: A Companion to Methods in Enzymology 15: 323-331(1998). Compounds are initially dissolved in either ethanol or in DMSOto provide a working stock solution. The synthetic substrate used in theassay isN-(4-methyl-7-coumarinyl)-N-α-(tert-butyloxy-carbonyl)-N-Ω-acetyllysineamide(MAL).

[0072] The assay is performed in a final total volume of 120 μLconsisting of 100 μL of 15 mM tris-HCl buffer at pH 7.9 and 0.25 mMEDTA, 10 mM NaCl, 10% glycerol, 10 mM mercaptoethanol and the enzyme.The assay is initiated upon the addition of 10 μL of a test compoundfollowed by the addition of a fluorescent-labeled lysine substrate toeach assay tube in an ice bath for 15 minutes. The tubes are transferredto a water bath at 37° C. for an additional 90 minutes.

[0073] An initial assay is performed to determine the range of activityof each test compound. The determination of IC₅₀ values is made from theresults of five dilutions in range according to the expected potency foreach test compound. Each assay is duplicated or triplicated.

[0074] Other embodiments are within the scope of the following claims.

What is claimed is:
 1. A compound having the formula (I):

wherein A is a cyclic moiety selected from the group consisting of C₃₋₁₄ cycloalkyl, 3-14 membered heterocycloalkyl, C₄₋₁₄ cycloalkenyl, 3-8 membered heterocycloalkenyl, aryl, and heteroaryl; the cyclic moiety being optionally substituted with alkyl, alkenyl, alkynyl, alkoxy, hydroxyl, hydroxylalkyl, halo, haloalkyl, amino, thio, alkylthio, arylthio, aralkylthio, acylthio, alkylcarbonyloxy, alkyloxycarbonyl, alkylcarbonyl, alkylsulfonylamino, aminosulfonyl, or alkylsulfonyl; or A is a saturated branched C₃₋₁₂ hydrocarbon chain or an unsaturated branched C₃₋₁₂ hydrocarbon chain optionally interrupted by —O—, —S—, —N(R^(a))—, —C(O)—, —N(R^(a))—SO₂—, —SO₂—N(R^(a))—, —N(R^(a))—C(O)—O—, —O—C(O)—N(R^(a))—, —N(R^(a))—C(O)—N(R^(b))—, —O—C(O)—, —C(O)—O—, —O—SO₂—, —SO₂—O—, or —O—C(O)—O—, where each of R^(a) and R^(b), independently, is hydrogen, alkyl, alkenyl, alkynyl, alkoxy, hydroxylalkyl, hydroxyl, or haloalkyl; each of the saturated and the unsaturated branched hydrocarbon chain being optionally substituted with alkyl, alkenyl, alkynyl, alkoxy, hydroxyl, hydroxylalkyl, halo, haloalkyl, amino, thio, alkylthio, arylthio, aralkylthio, acylthio, alkylcarbonyloxy, alkyloxycarbonyl, alkylcarbonyl, alkylsulfonylamino, aminosulfonyl, or alkylsulfonyl; each of Y¹ and Y², independently, is —O—, —S—, —N(R^(c))—, —N(R^(c))—C(O)—O—, —N(R^(c))—C(O)—, —C(O)—N(R^(c))—, —O—C(O)—N(R^(c))—, —N(R^(c))—C(O)—N(R^(d))—, —O—C(O)—O—, or a bond; each of R^(c) and R^(d), independently, being hydrogen, alkyl, alkenyl, alkynyl, alkoxy, hydroxylalkyl, hydroxyl, or haloalkyl; L is a straight C₃₋₁₂ hydrocarbon chain optionally containing at least one double bond, at least one triple bond, or at least one double bond and one triple bond; the hydrocarbon chain being optionally substituted with C₁₋₄ alkyl, C₂₋₄ alkenyl, C₂₋₄ alkynyl, C₁₋₄ alkoxy, hydroxyl, halo, amino, thio, alkylthio, arylthio, aralkylthio, acylthio, nitro, cyano, C₃₋₅ cycloalkyl, 3-5 membered heterocycloalkyl, monocyclic aryl, 5-6 membered heteroaryl, C₁₋₄ alkylcarbonyloxy, C₁₋₄ alkyloxycarbonyl, C₁₋₄ alkylcarbonyl, or formyl; and further being optionally interrupted by —O—, —N(R^(e))—, —N(R^(e))—C(O)—O—, —O—C(O)—N(R^(e))—, —N(R^(e))—C(O)—N(R^(f))—, or —O—C(O)—O—; each of R^(e) and R^(f), independently, being hydrogen, alkyl, alkenyl, alkynyl, alkoxy, hydroxylalkyl, hydroxyl, or haloalkyl; and X¹ is O or S; X² is a halogen; provided that when Y¹ and Y² are each a bond, L is a C₆₋₁₂ hydrocarbon chain containing at least one double bond at C1, C2, C3 or C5 of the hydrocarbon chain from C═X¹, at least one triple bond, or at least one double bond and one triple bond, the hydrocarbon chain being optionally substituted with C₁₋₄ alkyl, C₂₋₄ alkenyl, C₂₋₄ alkynyl, C₁₋₄ alkoxy, hydroxyl, halo, amino, thio, alkylthio, arylthio, aralkylthio, acylthio, nitro, cyano, C₃₋₅ cycloalkyl, 3-5 membered heterocycloalkyl, monocyclic aryl, 5-6 membered heteroaryl, C₁₋₄ alkylcarbonyloxy, C₁₋₄ alkyloxycarbonyl, C₁₋₄ alkylcarbonyl, or formyl; and further being optionally interrupted by —O—, —N(R^(e))—, —N(R^(e))—C(O)—O—, —O—C(O)—N(R^(e))—, —N(R^(e))—C(O)—N(R^(f))—, or —O—C(O)—O—; each of R^(e) and R^(f), independently, being hydrogen, alkyl, alkenyl, alkynyl, alkoxy, hydroxylalkyl, hydroxyl, or haloalkyl; or a salt thereof.
 2. The compound of claim 1, wherein X² is fluorine.
 3. The compound of claim 1, wherein Y¹ is not a bond.
 4. The compound of claim 1, wherein each of Y¹ and Y² independently, is —O—, —N(R^(c))—, or a bond.
 5. The compound of claim 1, wherein X¹ is O.
 6. The compound of claim 2, wherein X¹ is O.
 7. The compound of claim 6, wherein Y¹ is not a bond.
 8. The compound of claim 6, wherein each of Y¹ and Y², independently is —O—, —N(R^(c))—, or a bond.
 9. The compound of claim 1, wherein L is a C₆₋₈ hydrocarbon chain substituted with C₁₋₂ alkyl, C₁₋₂ alkoxy, hydroxyl, —NH₂, —NH(C₁₋₂ alkyl), or —N(C₁₋₂ alkyl)₂.
 10. The compound of claim 6, wherein L is a C₆₋₈ hydrocarbon chain substituted with C₁₋₂ alkyl, C₁₋₂ alkoxy, hydroxyl, —NH₂, —NH(C₁₋₂ alkyl), or —N(C₁₋₂ alkyl)₂.
 11. The compound of claim 1, wherein L is a C₄₋₁₂ hydrocarbon chain.
 12. The compound of claim 1, wherein L is a C₅₋₁₂ hydrocarbon chain.
 13. The compound of claim 1, wherein L is a C₅₋₁₀ hydrocarbon chain.
 14. The compound of claim 6, wherein L is a C₅₋₁₀ hydrocarbon chain.
 15. The compound of claim 1, wherein L is an unsaturated hydrocarbon chain containing at least two double bonds.
 16. The compound of claim 1, wherein A is a C₅₋₈ cycloalkenyl, 5-8 membered heteroalkenyl, phenyl, naphthyl, indanyl, or tetrahydronaphthyl optionally substituted with alkyl alkenyl, alkynyl, alkoxy, hydroxyl, hydroxylalkyl, halo, haloalkyl, or amino.
 17. The compound of claim 6, wherein A is a C₅₋₈ cycloalkenyl, 5-8 membered heteroalkenyl, phenyl, naphthyl, indanyl, or tetrahydronaphthyl optionally substituted with alkyl alkenyl, alkynyl, alkoxy, hydroxyl, hydroxylalkyl, halo, haloalkyl, or amino.
 18. The compound of claim 1, wherein A is phenyl, Y¹ is a bond, and L is a C₆₋₁₂ hydrocarbon chain containing three double bonds and the carbon adjacent to Y¹ is substituted with phenyl.
 19. The compound of claim 6, wherein A is phenyl, Y¹ is a bond, and L is a C₆₋₁₂ hydrocarbon chain containing three double bonds and the carbon adjacent to Y¹ is substituted with phenyl.
 20. The compound of claim 1, wherein A is phenyl, Y¹ is a bond, and L is a C₃₋₁₂ hydrocarbon chain and the carbon adjacent to Y¹ is substituted with two phenyl groups.
 21. The compound of claim 6, wherein A is phenyl, Y¹ is a bond, and L is a C₃₋₁₂ hydrocarbon chain and the carbon adjacent to Y¹ is substituted with two phenyl groups.
 22. The compound of claim 1, wherein the compound is 1,1,1-trifluoro-8-phenyl-3,5,7-octatrien-2-one or 1,1,1-trifluoro-8-phenoxy-3,5,7-octatrien-2-one.
 23. A method of inhibiting histone deacetylation activity in cells comprising contacting the cells with an effective amount of a compound containing a trihalomethylcarbonyl group, thereby treating one or more disorders mediated by histone deacetylase or stimulating hematopoietic cells ex vivo, and determining whether the level of acetylated histones in the treated cells is higher than in untreated cells under the same conditions.
 24. The method of claim 23, wherein the compound is of the formula (I):

wherein A is a cyclic moiety selected from the group consisting of C₃₋₁₄ cycloalkyl, 3-14 membered heterocycloalkyl, C₄₋₁₄ cycloalkenyl, 3-8 membered heterocycloalkenyl, aryl, and heteroaryl; the cyclic moiety being optionally substituted with alkyl, alkenyl, alkynyl, alkoxy, hydroxyl, hydroxylalkyl, halo, haloalkyl, amino, thio, alkylthio, arylthio, aralkylthio, acylthio, alkylcarbonyloxy, alkyloxycarbonyl, alkylcarbonyl, alkylsulfonylamino, aminosulfonyl, or alkylsulfonyl; or A is a saturated C₁₋₁₂ hydrocarbon chain or an unsaturated C₂₋₁₂ hydrocarbon chain optionally interrupted by —O—, —S—, —N(R^(a))—, —C(O)—, —N(R^(a)) SO₂—, —SO₂—N(R^(a))—, —N(R^(a))—C(O)—O—, —O—C(O)—N(R^(a))—, —N(R^(a))—C(O)—N(R^(b))—, —C(O)—N(R^(a))—, —N(R^(a))—C(O)—, —O—C(O)—, —C(O)—O—, —O—SO₂—, —SO₂—O—, or —O—C(O)—O—, where each of R^(a) and R^(b), independently, is hydrogen, alkyl, alkenyl, alkynyl, alkoxy, hydroxylalkyl, hydroxyl, or haloalkyl; each of the saturated and the unsaturated branched hydrocarbon chain being optionally substituted with alkyl, alkenyl, alkynyl, alkoxy, hydroxyl, hydroxylalkyl, halo, haloalkyl, amino, alkylcarbonyloxy, alkyloxycarbonyl, alkylcarbonyl, alkylsulfonylamino, aminosulfonyl, or alkylsulfonyl; each of Y¹ and Y², independently, is —CH₂—, —O—, —S—, —N(R^(c))—, —N(R^(c))—C(O)—O—, —C(O)—, —C(NOR^(c))—, —N(R^(c))—C(O)—, —C(O)—N(R^(c))—, —O—C(O)—N(R^(c))—, —N(R^(c))—C(O)—N(R^(d))—, —O—C(O)—O—, or a bond; each of R^(c) and R^(d), independently, being hydrogen, alkyl, alkenyl, alkynyl, alkoxy, hydroxylalkyl, hydroxyl, or haloalkyl; L is a straight C₁₋₁₂ hydrocarbon chain optionally containing at least one double bond, at least one triple bond, or at least one double bond and one triple bond; the hydrocarbon chain being optionally substituted with C₁₋₄ alkyl, C₂₋₄ alkenyl, C₂₋₄ alkynyl, C₁₋₄ alkoxy, hydroxyl, halo, amino, nitro, cyano, C₃₋₅ cycloalkyl, 3-5 membered heterocycloalkyl, monocyclic aryl, 5-6 membered heteroaryl, C₁₋₄ alkylcarbonyloxy, C₁₋₄ alkyloxycarbonyl, C₁₋₄ alkylcarbonyl, or formyl; and further being optionally interrupted by —O—, —N(R^(e))—, —N(R^(e))—C(O)—O—, —O—C(O)—N(R^(e))—, —N(R^(e))—C(O)—N(R^(f))—, or —O—C(O)—O—; each of R^(e) and R^(f), independently, being hydrogen, alkyl, alkenyl, alkynyl, alkoxy, hydroxylalkyl, hydroxyl, or haloalkyl; X¹ is O or S; and X² is a halogen; or a salt thereof.
 25. The method of claim 23, wherein the disorder is selected from the group consisting of cancer, hemoglobinopathies, thalassemia, sickle cell anemia, cystic fibrosis, protozoan infection, spinal muscular atrophy, Huntington's disease, alpha-1 anti-trypsin, retrovirus gene vector reactivation, wound healing, hair growth, peroxisome biogenesis disorder, and adrenoleukodystrophy.
 26. The method of claim 24, wherein the disorder is selected from the group consisting of cancer, hemoglobinopathies, thalassemia, sickle cell anemia, cystic fibrosis, protozoan infection, spinal muscular atrophy, Huntington's disease, alpha-1 anti-trypsin, retrovirus gene vector reactivation, wound healing, hair growth, peroxisome biogenesis disorder, and adrenoleukodystrophy.
 27. The method of claim 23, wherein the disorder is cancer, cystic fibrosis, or adrenoleukodystrophy.
 28. The method of claim 24, wherein the disorder is cancer, cystic fibrosis, or adrenoleukodystrophy.
 29. The method of claim 23, wherein hematopoietic cells are stimulated ex vivo.
 30. The method of claim 24, wherein hematopoietic cells are stimulated ex vivo.
 31. The method of claim 24, wherein the compound is 1,1,1-trifluoro-5-phenyl-2-pentanone, 1,1,1-trifluoro-8-phenyl-3,5,7-octatrien-2-one, or 1,1,1-trifluoro-8-phenoxy-3,5,7-octatrien-2-one. 